to the new linker has been shown to be both efficient and selective.
The chemical cleavage of the ligand plus its bioaffinity partner
from the gel has been shown to be comparable to a classical
cleavage process under denaturing conditions; and indeed the two
processes are both compatible with the new linker. Although the
use of this linker has been demonstrated using the agarose matrix
Affi-Gel 10, we believe that it will find widespread application on
a range of activated matrix and/or chip surfaces. It is anticipated
that the use of this azo linker as means of coupling biotin to the
ligand of interest will also allow an affinity-independent version
of the more classical manifold for target identification based
upon immobilisation on a (strept)avidin coated matrix, as well
as allowing in vivo experiments. Investigations with several ligands
of interest (whose propargyl derivatives retain the activity of the
lead compound) with known, or potential, affinity partners are
currently under progress in our research group.
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Acknowledgements
We thank the EC FP6 for funding (Marie Curie EST Fellowships
to FL and CMJ; Contract: MEST-CT-2005-020744) and Gareth
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Notes and references
§ Whilst this reduction has found widespread use in biochemical applica-
tions, the concomitant reduction of some amino acids such as nitrotyrosine
to aminotyrosine has been reported under these conditions.9b
¶ Synthetically this would require coupling of the amino group of the azo
linker 1 to NHS-biotin, followed by click coupling of the biotin-linker
conjugate to the ligand.
ꢀ The weak bands at 66-97 kDa are likely to be serum albumin and similar
prominent serum proteins.
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